eDNA testing from Cellmark

 

Frequently asked questions

General questions:

eDNA, or environmental DNA is genetic material given off by an organism into its environment. In the case of great crested newts, the eDNA samples are collected from bodies of water. The newt DNA is released into the water via shed skin cells, mucus, sperm, eggs, faeces and decomposing animals. Studies suggest that both the larval stage and adult stages are detectible using eDNA analysis (Rees et al, 2017; Buxton et al, 2017).
This DNA given off into the water can persist for a number of weeks, allowing us to test for its presence in water samples.

Natural England’s eDNA protocol has proved to be a rapid and effective way to test for the presence/absence of great crested newts.
Biggs et al. (2015) showed that when newts were present, eDNA was 99.3% effective at detecting newts in ponds where they were known to occur. No false positive were reported for this study and 8.7% of sites had false negative (newts were present but not detected) results. This compares to detection efficacy of 76% for bottle trapping, 75% for torch surveys and 44% for egg searches.
Biggs et al. (2015) Using eDNA to develop a national citizen science-based monitoring programme for the great crested newt (Triturus cristatus). Biological Conservation 183 (2015) 19–28.

At the moment our test only detects GCN.

No. At this time eDNA analysis can only generate presence/absence results.

A licensed great crested newt surveyor must collect the sample if the eDNA test is in support of a license application.

Natural England accepts eDNA survey results from samples collected between 15 April and 30 June each year.

We can perform testing year round.

Ordering & Delivery of Kits:

  • Next Day eDNA service - £330
  • 2 day turnaround - £280* Introductory offer £250
  • 5 day turnaround - £225* Introductory offer £195
  • 10 day turnaround - £165* Introductory offer £135

These are the prices for the kit itself (everything required to collect the water sample), delivery of the kit to your location, the analysis, and the return of the results. Sample return by courier can be arranged at the cost of £20* per courier trip - * excludes return for Next Day eDNA - this costs £35 per courier trip. All prices are ex VAT.

Discounts are available - details are shown on the relevant webpages. If you are looking for larger bulk orders then please call us direct to discuss.

You can pay upfront by credit card over the telephone when you place your order or we can supply you with an invoice. Our payment terms are 30 days.

For credit card orders a VAT receipt should have been sent to you at the time of ordering if you wish another copy to be sent please email us at eDNA@cellmark.co.uk and this will be re-issued to you.

Orders placed before 3pm can normally (subject to stock availability) be dispatched to you by our courier for delivery on the next working day. Please note, deliveries to Scotland can often take a little longer to arrive.

Orders can be adjusted or cancelled prior to the dispatch of kits (please call us on 0800 193 3362), however once kits have been dispatched your order will be subject to our refund policy - see our terms and conditions for details.

If your kits have not arrived as expected please contact us and we can check their progress. If you wish, when we book the courier, we can issue you with a tracking reference number.

Sampling and returning your samples

There is a risk of transferring eDNA on your boots, equipment etc. as you move from pond to pond. You should make sure that you never go in the water when sampling a pond. When possible take your eDNA samples before you do other surveys. Exceptionally muddy/wet conditions may also increase the chances of contamination due to splashing of GCN containing water or mud onto clothing/boots or equipment.

We recommend using chlorine based bleach to destroy any DNA on boots and equipment.

All pond sampling must be done following the Natural England technical advice note.
A copy of our sampling instructions is sent out with every kit.

Each kit will contain the following items:

  • A sterile 30 mL ladle,
  • A sterile self-supporting Whirl-Pak plastic bag with 1 L capacity,
  • A sterile 10 mL pipette to resample the pond water,
  • 6 sterile 50 mL centrifuge tubes containing preservative (Absolute Ethanol (200 Proof),
  • 2 pairs of sterile gloves,
  • 1 gripseal bag
  • 1 Mail-Tuff return envelope,
  • 1 sample return form and
  • 1 instruction sheet.
The sample tubes, packaging and sample return form have all been pre-barcoded for your convenience.

1 kit can be used for ponds up to 1 hectare in size.

For larger bodies of water, Natural England recommends using 1 kit per hectare. If multiple kits are required, we recommend that the circumference of the lake is divided into equal sections based on the number of kits to be used. One kit is then used to sample each of the sections.

The concentrations of GCN eDNA may be different in different regions of the pond. GCN may favor some areas of a pond more than others. Their eDNA is likely to be more concentrated in the more heavily populated areas. Sampling multiple areas increase the chances of collecting GCN DNA if they are present.

This is in accordance with the Natural England guidelines.

The DNA/cellular material gradually sinks in the water column and will often be present in larger amounts close to the pond bottom. Try to avoid collecting sediment. It can interfere with the analysis.

Small amounts of these materials do not affect the analysis. Avoid transferring larger debris to the 50 ml sample tubes. Try to avoid collecting bottom sediment

  • There is the potential for GCN DNA to remain in the sediment when there is no GCN activity in the water body during the current breeding season
  • Sediment can cause inhibition of the PCR analysis used to detect GCN eDNA which could lead to an inconclusive result.
  • Sediment can interfere with the DNA extraction procedure.

 

Please try to avoid including sediment in the sample as it can interfere with the laboratory testing processes. Upon sample receipt, we inspect the samples for the presence of sediment, algae, precipitates and any other debris. If samples contain excess amounts of debris we may have to fault the sample. In this instance we will contact the sampler to highlight the issue. On occasion this may require the submission of a new sample - a replacement kit will cost £25.

Upon sample receipt we inspect your samples for the presence of sediment, algae, precipitates and any other debris. Algae can make the DNA extraction more difficult to perform so it is helpful it can be avoided.

If some of the ethanol solution spills, add proportionately less of the pond sample to that tube. If a large amount of the ethanol has spilled or multiple tubes have spilled, please call us on 0800 193 3362 for advice.

Natural England’s technical advice note states that kits should be used within about two weeks of receipt. Ideally, you should return the kits as soon as possible after the sampling is completed.

Ideally, you should return the kits as soon as possible after the sampling is completed. However, you can return the kits to the laboratory within two weeks after pond water collection. If samples must be stored for a few days prior to returning them to the laboratory please store them in the refrigerator.

There are three options:

  1. We operate a 24/7 365 sample reception service, so if you’re local, you can drop it into our reception (Unit 16) in Abingdon, however for security reasons please phone in advance (during normal office hours) to let us know who will be dropping the sample off and an approximate time.
  2. Use a Cellmark courier. Call our customer services department on 0800 193 3362 and they will arrange a courier and email you the correct labels for you to attach to the outer packaging. Our standard courier charge is £20 however, this rises to £35 if you are using our Next Day testing service.
  3. Using your own courier. Please contact us to confirm your choice so we can plan for your sample’s arrival.

Unfortunately if you have ordered more kits than you require we will be unable to refund these unless the order is amended prior to the kits being sent out to you (in this case please email eDNA@cellmark.co.uk for advice). If you wish to return the unused kits to us for disposal then please return them to our laboratories alongside your pond samples.

You should never reuse any parts of the kit. Once you have used your ladle, gloves, Whirl-Pak bag and pipette, these must be disposed of. The risk of contamination is too high for them to be reused.

The samples should be okay at ambient temperature for a few days and they can be stored for up to 1 month at 4°C prior to laboratory analysis.

The ethanol in the 50 ml tubes helps to preserve the DNA. Refrigeration also slows the degradation process. DNA degradation will be faster at ambient temperature but should not have a large effect on sample outcome. It may be advisable to refrigerate the sample for a few days in excessively hot weather.

Analysis of samples

    The samples are processed as follows:
  • The DNA is extracted from the sample
  • The DNA extract is tested for PCR inhibition using a real-time PCR assay. If inhibition is found the sample is diluted prior to testing for the presence of great crested newts.
  • The sample is tested for degradation using a real-time PCR assay.
  • The sample is tested for presence of GCN DNA using a real-time PCR assay.

According to the Natural England technical advice note once collected, the samples can be stored for 1 month in the refrigerator prior to analysis. If possible, samples should be stored refrigerated before they are returned to us.

Reports

  • A positive result means that great crested newts are present in the water or have been present in the water in the recently.
  • A negative result indicates that DNA from the great crested newt has not been detected in the sample.
  • An inconclusive result can be issued for a couple of reasons.
    • DNA from the great crested newt has not been detected but the controls have indicated that the sample has been degraded.
    • DNA from the great crested newt has not been detected but the controls have indicated that the PCR has been inhibited.

    An inconclusive result may be due to the water chemistry, sediment or other debris in the sample. The pond could be retested (a new sample would be required) but if the water chemistry was the cause of the inconclusive result - the re-test would also be inconclusive.

If degradation (the results from the degradation control assay were outside of accepted limits) or inhibition (something in the DNA extract prevented PCR amplification) were observed, negative results will be reported as inconclusive.